The CHO cell line has been widely used for the production of target proteins such as recombinant antibody, human interferon-gamma (Goldman, et al., 1997, Cytotechnology, 23, 103-111), factor VIII (Ganne and Mignot, 1991, Cytotechnology, 6, 233-40), and thrombopoietin (Kim, et al., 2000, Biotechnol. Prog., 16, 775-781). For the production of target proteins in recombinant CHO cell cultures, production speed rate indicated the amounts of produced proteins per time unit from the live cells is important. In order to enhance the production speed rate, sodium butyrate (NaBu), which is known to enhance productivity by inducing transcription of foreign gene, has been used (Palermo, et al., 1991, J. Biotechnol., 19, 35-47). Despite the positive effect of sodium butyrate on protein production, there is a limitation in its application at a relatively high concentration to recombinant CHO cell culture for foreign protein production, since sodium butyrate can also inhibit cell growth, followed by rapid induction of apoptotic cell death of CHO cells. To fully exploit the beneficial effect of sodium butyrate on foreign protein production, a strategy of overcoming its cytotoxic effect has been attempted by the overexpression of survival proteins like Bcl-2 in CHO cells (Tey, et al., 2000, Biotechnol. Bioeng., 68, 31-43). Overexpression of Bcl-2 extended the culture longevity of recombinant CHO cells during batch culture by suppressing the NaBu-induced apoptosis and thereby resulted in doubling the final antibody concentration (Kim and Lee, 2000, Biotechnol. Bioeng., 71, 184-193). Although the overexpression of survival protein can endow the cells with more robustness in various culture modes and sub-optimal culture conditions (Cotter and Al-Rubeai, 1995, TIBTECH, 13, 150-155), most survival proteins have oncogenic properties. The oncogenic property may give rise to regulatory problems in the commercial production of therapeutic proteins. Thus, it may be necessary to screen potent apoptosis inhibitors other than survival proteins.
The cellular pathway leading to apoptosis involves the activation of members of a family of protease, caspases. To date, fourteen members of the caspase family have been identified in vertebrates, and at least eight are known to be involved in apoptotic cell death (Saunders, et al., 2000, Anal. Biochem., 284, 114-124). Among the various apoptosis-related caspases, extensive research has been performed on caspase-3, because caspase-3 has a broad range of intracellular protein substrates (Han, et al., 1997, J. Biol. Chem., 272, 13432-13436). Previously, the present inventors demonstrated that significant elevation in caspase-3 activity preceded the apparent physiological apoptotic progress induced by sodium butyrate and that the overexpression of Bcl-2 inhibited the NaBu-induced apoptosis by suppressing the activation of caspase-3 (Kim and Lee, 2000, Biotechnol. Bioeng., 71, 184-193). There are some reports that apoptosis is blocked successfully in NS0 and CHO cells by inhibiting the activity of caspase-3 or caspase-3-like proteases. At this time, peptide caspase inhibitor (McKenna and Cotter, 2000, Biotechnol. Bioeng., 67, 165-176) or metal cadmium (Yuan, et al., 2000, Toxicol. Appl. Pharmacol., 164, 321-329) is used to inhibit the caspase-3 activity. The use of peptide and metallic inhibitors of apoptosis, however, is unfavorable in large-scale commercial production of recombinant protein because of the high cost of peptide inhibitor and the carcinogenic properties of metallic inhibitor.
The antisense technology may be an alternative to suppress NaBu-induced apoptosis in recombinant CHO cells. Antisense strategy can be performed as transcription of antisense full sequence or partial fragment of target gene, or as introduction of an exogenous antisense oligonucleotide. Despite the time-consuming, laborious process in specific gene knockout, the transcription of antisense sequence is preferred to the introduction of exogenous antisense oligonucleotides because of its efficiency and low cost (Prati, et al., 1998, Biotechnol. Bioeng., 59, 445-450). Furthermore, although depending on both the sense and antisense sequences, the expression of antisense RNA of caspase seems to be able to achieve complete inhibition of apoptotic cell death at a low level of expression of antisense RNA, removing the negative effect on growth rate and productivity originated from the synthesis of large quantities of survival protein (Singh, et al., 1997, Cytotechnology, 23, 87-93).
Thus, the present inventors adopted the antisense technology to suppress the NaBu-induced apoptosis. The present invention has been accomplished by confirming that apoptosis caused by sodium butyrate is suppressed when the expression vector inducing antisense RNA transcription for caspase-3 is introduced to the recombinant CHO cells producing a humanized antibody specific to S-surface antigen of hepatitis B virus.